Patronin governs minus-end-out orientation of dendritic microtubules to promote dendrite pruning in Drosophila

  1. Yan Wang
  2. Menglong Rui
  3. Quan Tang
  4. Shufeng Bu
  5. Fengwei Yu  Is a corresponding author
  1. National University of Singapore, Singapore
8 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Patronin is required for dendrite pruning in ddaC neurons.

(A) A schematic representation of dendrite pruning in ddaC neurons. (B–F) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. While the wild-type neurons …

https://doi.org/10.7554/eLife.39964.002
Figure 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.005
Figure 1—figure supplement 1
Patronin is required for dendrite pruning and arborization in sensory neurons.

(A–E) Live confocal images of da neurons expressing mCD8-GFP at w3L, WP, 16 hr and 20 hr, 32 hr APF. (A) While the control neurons pruned all the dendrites at 16 hr APF, ddaC neurons overexpressing p…

https://doi.org/10.7554/eLife.39964.003
Figure 1—figure supplement 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.004
Figure 2 with 1 supplement
Overexpression of Patronin causes dendrite pruning defects in ddaC neurons.

(A–E) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4, Gal44-77 or ppk-CD4-tdGFP at WP and 16 hr APF. While ddaC neurons overexpressing the UAS control construct pruned …

https://doi.org/10.7554/eLife.39964.006
Figure 2—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.009
Figure 2—figure supplement 1
Overexpression of Patronin causes dendrite arborization defects in ddaC neurons.

(A) Confocal images of ddaC neurons expressing mCD8-GFP and immunostained for anti-Patronin at w3L stage. ddaC somas are marked by dashed lines, and axons by arrows. Endogenous Patronin was detected …

https://doi.org/10.7554/eLife.39964.007
Figure 2—figure supplement 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.008
Figure 3 with 1 supplement
The CKK domain is important for Patronin to govern dendrite pruning.

(A–D, G–J) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 or Gal44-77 at WP and 16 hr APF. Low-level expressions of PatroninΔCH (B) and PatroninCKK (D), but not PatroninΔ…

https://doi.org/10.7554/eLife.39964.010
Figure 3—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.013
Figure 3—figure supplement 1
Structure-function analysis of the Patronin protein.

(A) A schematic representation of full-length and truncated Patronin proteins and a summary of structure-function analysis of Patronin. Patronin contains multiple domains: a calponin homology (CH) …

https://doi.org/10.7554/eLife.39964.011
Figure 3—figure supplement 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.012
Figure 4 with 1 supplement
Patronin is required for proper distribution of dendritic and axonal MT markers.

(A–C, F–H) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-β-gal or Kin-β-gal and immunostained for β-galactosidase at wL3 stage. ddaC somas are marked by asterisks, axons by arrows and …

https://doi.org/10.7554/eLife.39964.014
Figure 4—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.016
Figure 4—figure supplement 1
Patronin is required for proper distribution of the MT marker Nod-β-gal in ddaC dendrites.

(A) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-β-gal and immunostained for β-galactosidase at wL3 stage. Nod-β-gal levels were reduced in the dendrites but enriched in the somas of patr…

https://doi.org/10.7554/eLife.39964.015
Figure 5 with 1 supplement
Patronin is required for the minus-end-out orientation of dendritic MTs in ddaC neurons.

(A–B, G–H) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In control RNAi (A) or UAS control (G) ddaC neurons, dendritic EB1-GFP comets predominantly moved …

https://doi.org/10.7554/eLife.39964.017
Figure 5—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.020
Figure 5—figure supplement 1
Patronin is required for dendritic and axonal MT orientations in ddaC neurons.

(A) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In control RNAi ddaC neurons, dendritic EB1-GFP comets predominantly moved retrogradely towards the somas. …

https://doi.org/10.7554/eLife.39964.018
Figure 5—figure supplement 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.019
Figure 6 with 1 supplement
Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotypes in MT orientation in ddaC dendrites.

(A, B) Representative kymographs of EB1-GFP comets in control or mutant ddaC dendrites. In patronin RNAi #1 and control RNAi co-expressing ddaC neurons (A), dendritic EB1-GFP comets moved …

https://doi.org/10.7554/eLife.39964.021
Figure 6—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.024
Figure 6—figure supplement 1
Attenuation of Klp10A, a kinesin-13 MT depolymerase, suppresses the patronin phenotype in MT orientation in ddaC dendrites.

(A) Quantification of the percentage of dendrite branches showing different levels of anterograde EB1-GFP comets in the control and mutant ddaC neurons. (B) Representative kymographs of EB1-GFP …

https://doi.org/10.7554/eLife.39964.022
Figure 6—figure supplement 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.023
Figure 7 with 1 supplement
Patronin promotes dendrite pruning by orienting uniform minus-end-out MT arrays in dendrites.

(A–C, F–H) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. Red arrowheads point to the ddaC somas. (A–C) ddaC neurons co-expressing patronin RNAi #1 …

https://doi.org/10.7554/eLife.39964.025
Figure 7—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.028
Figure 7—figure supplement 1
patronin appears not to genetically interact with kat-60, kat-60L1 or tau in dendrite pruning.

(A–E) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at 16 hr APF. Similar to the control neurons, klp10A RNAi (#1 or #2) or kat-60 RNAi (#1 or #2) ddaC neurons pruned …

https://doi.org/10.7554/eLife.39964.026
Figure 7—figure supplement 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.027
Figure 8 with 3 supplements
Klp10A overexpression phenocopies patronin knockdown in dendrite pruning and dendritic MT orientation.

(A–C) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at WP and 16 hr APF. Red arrowheads point to the ddaC somas. ddaC neurons overexpressing the UAS control construct …

https://doi.org/10.7554/eLife.39964.029
Figure 8—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.036
Figure 8—figure supplement 1
Overexpression of Klp10A causes dendritic MT orientation defects in ddaC neurons.

(A–B) Confocal images of ddaC neurons expressing mCD8-GFP, Nod-lacZ or Kin-lacZ and immunostained for β-galactosidase at wL3 stage. ddaC somas are marked by asterisks, axons by arrows and dendrites …

https://doi.org/10.7554/eLife.39964.030
Figure 8—figure supplement 1—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.031
Figure 8—figure supplement 2
The CKK domain of Patronin is important for dendrite pruning and dendritic MT orientation.

(A, D) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at 16 hr APF. Low-level expression of PatroninCKK under the control of UASp promoter significantly rescued the …

https://doi.org/10.7554/eLife.39964.032
Figure 8—figure supplement 2—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.033
Figure 8—figure supplement 3
CNN and APC1/2 appear to be required for dendrite pruning of ddaC neurons.

(A–D) Live confocal images of ddaC neurons expressing mCD8-GFP driven by ppk-Gal4 at 16 hr APF. (A–B) While the control neurons pruned all the dendrites, cnnhk21 neurons (A), or APC2N175K, APC1Q8

https://doi.org/10.7554/eLife.39964.034
Figure 8—figure supplement 3—source data 1

Extended statistical data as Microsoft Excel spreadsheet.

https://doi.org/10.7554/eLife.39964.035

Tables

Key resources table
Reagent typeDesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent
(D. melangoaster)
UAS-MicalN-terother(Terman et al., 2002)
Genetic reagent
(D. melangoaster)
SOP-flp (#42)other(Matsubara et al., 2011)
Genetic reagent
(D. melangoaster)
ppk-Gal4 on II and
III chromosome
other(Grueber et al., 2003)
Genetic reagent
(D. melangoaster)
UAS-Kin-β-galother(Clark et al., 1997)
Genetic reagent
(D. melangoaster)
UAS-EB1-GFPother(Stone et al., 2008)
Genetic reagent
(D. melangoaster)
UASp-mCherry-Patroninother(Nashchekin et al., 2016)
Genetic reagent
(D. melangoaster)
patroninc9-c5other(Nashchekin et al., 2016)
Genetic reagent
(D. melangoaster)
UAS-GFP-Patroninother(Derivery et al., 2015)
Genetic reagent
(D. melangoaster)
UASp-Arf79F-EGFPother(Shao et al., 2010)
Genetic reagent
(D. melangoaster)
UAS-Klp10Athis paper
Genetic reagent
(D. melangoaster)
UAS-GFP-Klp10Athis paper
Genetic reagent
(D. melangoaster)
UAS-Patroninthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-Patroninthis paper
Genetic reagent
(D. melangoaster)
UASp-PatroninΔCHthis paper
Genetic reagent
(D. melangoaster)
UASp-PatroninΔCKKthis paper
Genetic reagent
(D. melangoaster)
UASp-PatroninCKKthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninΔCHthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninΔCKKthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninCC1-3this paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninCKKthis paper
Genetic reagent
(D. melangoaster)
UAS-Venus-PatroninCHthis paper
Genetic reagent
(D. melangoaster)
Gal4109(2)80Bloomington
Stock Center
BDSC: 8769
Genetic reagent
(D. melangoaster)
ppk-CD4-tdGFPBloomington
Stock Center
BDSC: 35843
Genetic reagent
(D. melangoaster)
GSG2295-Gal4Bloomington
Stock Center
BDSC: 40266
Genetic reagent
(D. melangoaster)
Gal44-77Bloomington
Stock Center
BDSC: 8737
Genetic reagent
(D. melangoaster)
UAS-Nod-β-galBloomington
Stock Center
BDSC: 9912
Genetic reagent
(D. melangoaster)
patronin RNAi #2Bloomington
Stock Center
BDSC: 36659
Genetic reagent
(D. melangoaster)
klp10A RNAi #2Bloomington
Stock Center
BDSC: 33963
Genetic reagent
(D. melangoaster)
Gal42-21Bloomington
Stock Center
FBal0328157
Genetic reagent
(D. melangoaster)
patronin RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v108927
Genetic reagent
(D. melangoaster)
klp10A RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v41534
Genetic reagent
(D. melangoaster)
control RNAiVienna Drosophila
RNAi Centre
VDRC: v36355
Genetic reagent
(D. melangoaster)
control RNAiVienna Drosophila
RNAi Centre
VDRC: v37288
Genetic reagent
(D. melangoaster)
patronink07433Drosophila Genetic
Resource Center
#111217
Genetic reagent
(D. melangoaster)
patronin RNAi #3National Institute
of Genetics, Japan
#18462 Ra-1
Genetic reagent
(D. melangoaster)
kat-60 RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v38368
Genetic reagent
(D. melangoaster)
kat-60 RNAi # 2Vienna Drosophila
RNAi Centre
VDRC: v106487
Genetic reagent
(D. melangoaster)
kat-60L1 RNAi # 1Vienna Drosophila
RNAi Centre
VDRC: v31599
Genetic reagent
(D. melangoaster)
kat60-L1 RNAi # 2Vienna Drosophila
RNAi Centre
VDRC: v108168
Genetic reagent
(D. melangoaster)
tau RNAi # 1Bloomington
Stock Center
BDSC: 28891
Genetic reagent
(D. melangoaster)
tau RNAi # 2Bloomington
Stock Center
BDSC: 40875
Genetic reagent
(D. melangoaster)
cnnhk21Bloomington
Stock Center
BDSC: 5039
Genetic reagent
(D. melangoaster)
FRT 82B, APC2N175K,
APC1Q8
Bloomington
Stock Center
BDSC: 7211
Genetic reagent
(D. melangoaster)
futschN94Bloomington
Stock Center
BDSC: 8805
Antibodyanti-β-galactosidasePromegaCat#: Z37831:1000
Antibodyanti-PatroninM Gonzalez-Gaitan1:500
AntibodyCy3-conjugated goat
anti-Rabbit antibody
JacksonCat#111-165-0031:500
Antibody647-conjugated goat
anti-Rabbit antibody
JacksonCat#111-605-1441:500
Antibodyanti-FutschDSHB22c101:50

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